Advance Journal of Food Science and Technology 5(6): 783-786, 2013
ISSN: 2042-4868; e-ISSN: 2042-4876
© Maxwell Scientific Organization, 2013
Submitted: February 25, 2013
Accepted: March 27, 2013
Published: June 05, 2013
Preparation and Enzyme-Linked Immunosorbent Assay of Monoclonal Antibody
Against Ampicillin
1
Yan Lu, 2Xuping Zhou, 3Hongyu Zhao, 4 Jian Sun and 1Guojuan Wu
College of Animal Science and Technology, Beijing University of Agriculture,
Beijing 102206, P.R. China
2
College of Urban and Rural Development, Beijing University of Agriculture, Beijing 102206, P.R. China
3
National Institute of Biological Sciences, Beijing 100101, P.R. China
4
Department of Animal Husbandry and Veterinary Medicine, Beijing Vocational College Agriculture,
Beijing 102442, P.R. China
1
Abstract: Ampicillin (AMP) was coupled with mcKLH by the method of EDC intermediate and injected into
Balb/c mice as immunogen. The hybridomas were obtained by fusing mouse myeloma cells SP2/0 with splenocytes
from the mice immunized with AMP-KLH. The chromosomal numbers were 97-104, the results indicated that the
subclass of the McAb was IgG2a, the titer of ascitic fluid was 2×106, the affinity constant was 3×10-10 mol/L, the
molecular weight was 154KD. The cross-reactivities of McAb to other antibiotics were below 0.01%. The linear
detecting range of calibration curves to detect AMP was 0.5 ng/mL-100 ng/mL. The formular was Y =
0.0569X+0.1308, R2 = 0.9948. The limit of detection was 0.3 ng/mL.
Keywords: Ampicillin, ELISA, monoclonal antibody
2001) and penicillin receptor protein test (Setford et al.,
1999). However, these methods have not been widely
used despite they meet the requirement in terms of
maximum residue limits (MRL). In the present study,
hybridoma technology was employed to establish
hybridoma cell lines capable of producing monoclonal
Antibody Against Ampicillin (AMP) and a rapid AMP
detection technology was developed via EnzymeLinked Immunosorbent Assay (ELISA).
INTRODUCTION
Penicillin is one of the oldest antimicrobial agents
widely used for controlling of cow mastitis and
treatment of urinary tract, reproductive tract,
gastrointestinal tract and respiratory infections. Due to
specific side effects related to allergic reaction and drug
resistance, the use of penicillin to animals and the
penicillin residues in animal-based food are strictly
controlled and monitored in many countries. A variety
of analytical methods have been developed for
penicillin residue assays, including the initial microbial
growth inhibition test and the relative sensitive
instrumental analysis method (Lih et al., 1996;
Sorensen et al., 1997; Fontes et al., 1996). At present,
the high-performance liquid chromatography (HPLC),
immunoassay and microbioassay are frequently used
for penicillin residue assays. Of these, the HPLC is
sensitive and highly efficient, thus is commonly used at
a global scale. However, this method requires special
detection equipment. By comparison, microbioassay is
easy to operate but its sensitivity is relatively low.
Immunoassay is a simple, rapid, sensitive analytical
method with broad application prospects, which
combines the antigen-antibody reaction with sensitive
detection system (Li et al., 2002). In addition, several
new detection methods have been developed in recent
years, such as capillary electrophoresis (Ahrer et al.,
MATERIALS AND METHODS
The AMP standard, Bovine Serum Albumin
(BSA), Freund's adjuvant, O-Phenylenediamine (OPD),
PEG solution, horseradish peroxidase (HRP)-labeled
goat anti-mouse IgG, HAT and HT solutions and the
IgG subclass reagents were purchased from Sigma;
low-glucose dry-powdered DMEM and high-quality
fetal bovine serum were purchased from Hyclone; EDC
coupling kit was purchased from Pierce; Balb/c mice
were purchased from the Laboratory Animal Center of
the Chinese Academy of Military Medical Sciences;
and SP2/0 myeloma cells were stored in the High-tech
Laboratory of the Institute of Animal Husbandry and
Veterinary Medicine, Beijing Academy of Agriculture
and Forestry Sciences.
Methods:
Corresponding Author: Guojuan Wu, College of Animal Science and Technology, Beijing University of Agriculture, Bei Nong
Road No. 7, Beijing 102206, P.R. China, Tel.: +86 010 80796702
783
Adv. J. Food Sci. Technol., 5(6): 783-786, 2013
Synthesis and animal immunity assay of AMPholoantigen: Synthesis of AMP-BSA and AMP-KLH:
The AMP was coupled with BSA and KLH using the
EDC method. The obtained AMP-KLH was used as the
immunogen and AMP-BSA as the coating antigen.
Intraperitoneal injection was used for animal immunity
assay and cell fusion was performed on the spleen of
selected mice with high serum titer.
separating gel. After electrophoresis, the gels were
stained and bleached for observation.
Cross-reaction with β-lactams and other common
antibiotics: The working solutions of tested drugs were
prepared at the concentration of 100μg, 10μg, 1μg and
0.1 μg/mL. The concentration-inhibition rate curve was
established and used to calculate the cross-reaction
rate:Cross-reaction rate = (AMP IC 50 /Test drug
IC 50 )×100% (IC 50 is the inhibition rate of 50%)
Preparation and screening of hybridoma cells:
Myeloma cells (1×107 cells) and immune cells from
mouse spleen (1×108 cells) were harvested at the
logarithmic phase and fused with 50% PEG according
to the conventional method. The supernatant was
cultured by indirect ELISA and strong positive clones
were chosen. The cells were cloned for 3 times by
limited dilution and the hybridomas with a positive rate
of 100% were further cultured and frozen in liquid
nitrogen.
Identification of immunoglobulin class and subclass:
The immunoglobulin class and subclass were identified
using the Sigma antibody subclass identification kit
following the manufacture’s instruction.
Determination of affinity constants: The antigencoated microtiter plates diluted to 2, 1 and 0.5 μg/mL
were transferred to separate wells (100 μL each) for
incubation (overnight, 4 °C). After sealed with 1% BSA
at 37 °C for 1 h, the microtiter plates was added with
serial dilution of monoclonal antibodies with known
concentrations and incubated at 37 °C for 1 h.
Thereafter, the enzyme-labeled secondary antibody was
added at an appropriate concentration and the OPD
substrate buffer was used for color reaction. The
reaction was terminated by 2 mol/L H 2 SO 4 and the OD
value of each well was measured at 490 nm. Three
measurement curves were made using the
concentrations of monoclonal antibody as the abscissa
and the corresponding OD values as the ordinate. The
concentration of monoclonal antibody corresponding to
the OD value of 50%, i.e., [Ab]t, was obtained based on
that corresponding to the OD of 100% (the plain part
near the top of each line). Accordingly, the 3 values
[Ab]t, [Ab']t and [Ab"]t were obtained and the K value
was calculated as follows: K1 = 1/2(2[Ab′]t-[Ab]t), K2
= 1/2([Ab″]t -[Ab′]t) and K3 = 3/2([Ab″]t -[Ab]t. The
average value of K1, K2 and K3 were taken as the
affinity constant.
Characterization of anti-AMP-McAb: The McAb
supernatant (or McAb ascites) of cell culture was
consecutively diluted from 1000-fold using the
doubling dilution method. To the diluted McAb
supernatant was added the microtiter plates coated with
AMP-BSA, followed by HRP-conjugated goat antimouse IgG. Two wells were established for the positive
control (positive mouse serum), negative control (nonimmune mouse serum) and Blank Control (PBS). The
test result was considered to be positive when the OD
value at 490 nm was greater than or twice that of the
negative control. The titer was taken as the highest
dilution of the positive wells.
The hybridomas in the logarithmic phase were
added with 0.1 μg/mL colchicine and then cultured for
6-10 h at 37ºC. Then, the cells were collected,
centrifuged at 200g for 10min, treated with 5 mL of
0.075 mol/L KCl at 37 ºC and fixed with acetic acid
and methanol (1:3, v/v) on a glass slide for three times.
The slide was air-dried, stained with 10% Giemsa for
10 min, washed with distilled water and then naturally
dried. One-hundred complete intermediate nucleated
cells were examined by light microscopy and the
number of the chromosomes was recorded.
Preparation of standard curve: The standard curve
was prepared using the indirect competitive ELISA
method. The standard was doubly diluted to 0.01–100
ng/ml. The obtained OD value without AMP inhibition
was B0 and that with AMP inhibition was B. The
standard curve was prepared using the inhibition rate
[(B0-B)/B0] as the coordinate and the negative
logarithm of the AMP concentration as the abscissa.
The regression equation was established for the
standard curve.
Cross-reaction with KLH and BSA: The cells on
microtiter plates coated with AMP-BSA, KLH or BSA
were sealed with PBS containing 5% skim milk powder
and then added with diluted monoclonal antibody. After
the reaction, HRP-conjugated goat anti-mouse IgG was
added. The color reaction was achieved using an OPD
chromogenic substrate system and the OD value of
microtiter plates was determined at 490 nm.
Molecular weight determination. Purified ascites
were diluted to an appropriate range and
electrophoresized with polyacrylamide gel and 10%
Detection of AMP in commercial pure milk: The
AMP was added to the commercially pure fresh milk to
a certain concentration (0-100 ng/mL), oscillated at 4°C
overnight and centrifuged at 5000 rmp for 10 min. The
supernatant was collected and transferred to the
784
Adv. J. Food Sci. Technol., 5(6): 783-786, 2013
microtiter plate (100 μL per well). The standard curve
was prepared using the indirect competitive ELISA
method and the detection limit of AMP in milk was
calculated and.
RESULTS AND DISCUSSION
The fusion rate of cell colony was 99% three days
after fusion. Subsequent subcloning for 3 times
screened 8 hybridomas with high specificity and high
affinity. These hybridoma stably secreted antibody after
several passages, cryopreservation and recovery.
The titers of ascites were up to 2×106, significantly
higher than that of the cell supernatant (1:6400). The
concentration of purified monoclonal antibody of
ascites was 2 mg/mL and that of the cell supernatant
was 1.5 mg/mL after salting out and crude extraction
with saturated ammonium sulfate. The titer of purified
cell supernatant was 1:102400. Of the 8 hybridomas,
3D12 was relatively stable in terms of secretion of
antibodies and titer. Therefore, the 3D12 hybridoma
lines were selected for subsequent experiment.
The chromosome numbers of known mouse spleen
cells and SP2/0 myeloma cells were 40 and 60-70,
respectively, whereas that of hybridoma obtained in this
study was 97-104 (Fig. 1).
The molecular weight of the purified monoclonal
antibody was measured by SDS-PAGE electrophoresis.
According to the linear relationship between the
molecular weight and the migration rate, the molecular
weights of the light chain and the heavy chain of
monoclonal antibody were 26 kd and 51 kd,
respectively and the molecular weight of the
monoclonal antibody was 154 kd (Fig. 2). The subtype
of 3D12 strain was IgG2a.
Cross-reaction of McAb to other drugs (Table 1).
The protein concentration of the monoclonal antibody
was 1.5 mg/mL. According to the formula K=3×1010
mol/L, the concentrations of its dilutions were 1×10-3,
5×10-4, 2.5×10-4, 1×10-4, 5×10-5, 2.5×10-5, 1×10-5 and
5×10-6 mg/mL, respectively (Fig. 3).
The regression equation showed a good linear
relationship between the concentration range of 0.5-100
ng/mL. The linear regression equation between
inhibition rate and concentration (-logC, 1 mg/mL
mother liquor) was Y = 0.0569 X +0.1308 and the
correlation coefficient was R2 = 0.9948. Based on the
concentration corresponding to 10% inhibition rate, the
detection limit of AMP was calculated to be 0.3 ng/mL.
As for AMP added to the commercially-available,
pasteurized pure milk, the minimum detection limit was
0.4 ng/mL (Fig. 4).
Immunoassay, as an analytical technique
developed in recent years, has been widely used in
residue analysis of veterinary drugs, pesticides and
other small molecule compounds. Antibody is the core
reagent of immunoassay technology and its quality
greatly affects the standardization of the detection
methods (Wang and Li, 2000). Although the polyclonal
(a)
(b)
Fig. 1: The number of hybridism cell chromosome; (a)
Chromosome of hybridism cell; (b) Chromosome of
SP2/0
Fig. 2: The SDS-PAGE of monoclonal antibody against
ampicillin
1 is the octanoic acid-ammonium sulfate purification
method of antibody; 2 is unpurified mice ascites; M is
Mark of molecular weight
Table 1: The cross reaction rate of monoclonal antibodies to
antibiotics
Antibacterial drugs
Cross reaction rate (%)
Ampicillin
100
Ampicillin sodium
36
Penicillin sodium
< 0.007
Cefradine
< 0.001
Ceftiofur
< 0.001
Kanamycin
< 0.001
Streptomycin
< 0.001
Sulfadiazine
< 0.001
Gentamicin
< 0.001
antibody prepared by the application of small molecule
haptens and carrier protein conjugate as the immunized
animal contains an antibody against small molecules, it
has two disadvantages:
•
•
785
The low binding specificity of the polyclonal
antibodies produced by the conjugates and small
molecule antigen often leads to strong cross
reaction and causes false-positive results of
immunoassay.
The carrier protein has many B cell epitopes, which
stimulate the body to produce a large number of
antibodies against the carrier protein, thus reducing
the number of antibodies against small molecules
and the affinity. By comparison, the monoclonal
antibody is advantageous in terms of high
specificity and easy control and standardization.
Therefore, it has broader application prospects than
Adv. J. Food Sci. Technol., 5(6): 783-786, 2013
study, the working fluids were screened to ensure the
compliance of the antigen-antibody reaction.
The monoclonal antibody against AMP prepared in
the present study was characterized with high titration,
specificity and stability. The detection limit of the
standard curve prepared for AMP detection was lower
than the national maximum residue limit of ampicillin
in animal food as required. Further test of the ELISA
kit and test strip for AMP residue detection using this
antibody is ongoing.
ACKNOWLEDGMENT
Funding: This study was supported by the National
Natural Science Foundation of China (31201949), the
National Natural Science Foundation of China
(31172362); it was also supported by the Scientific
Research Improvement Foundation of Beijing
University of Agriculture (GJB2012003).
Fig. 3: The affinity constant of monoclonal antibody against
Ampicillin
REFERENCES
Ahrer, W., E. Scherwenk and W. Buchberger, 2001.
Determination of drug residues in water by the
combination of liquid chromatography or capillary
electrophoresis with electro spray mass
spectrometry. J. Chromatogr. A., 910(1): 69-78.
Fontes, E.M., S. Martins and B. Carrapico, 1996.
Determination of penicillin-G in commercialized
milk by reverse phase high performance liquid
chromatography. Toxicol. Lett., 88: 85.
Li, J.S., Y.M. Qiu and C. Wang, 2002. Analysis on the
Residues of Veterinary Drugs. Shanghai Scientific
and Technical Publishers, Shanghai, 2: 308-309.
Lih, S., A. Rehorek and M. Petz, 1996. Highperformance liquid chromatographic determination
of penicillins by means of automated solid-phase
extraction and photochemical degradation with
electrochemical detection. J. Chromatogr. A.,
729(2): 229-235.
Setford, S.J., R.M. Van Es, Y.J. Blandwater and S.
Kroger, 1999. Receptor binding protein
amperometric affinity sensor for rapid β-lactam
quantification in milk. Anal. Chem. Acta, 398(1):
13-22.
Sorensen, L.K., B.M. Rasmussen, J.O. Boison and K.
Lily, 1997. Simultaneous determination of six
penicillins in cows’raw milk by a multiresidue
high-performance liquid chromatographic method.
J. Chromatogr. B: Biomed. Sci. Appl., 694(2):
383-391.
Tan, Y. and D. Shi, 2000. Preparation and
characterization of monoclonal antibody to
chloramphenicol. Chinese J. Prev. Vet. Sci., 9: 22.
Wang, C. and S.J. Li, 2000. Determination of 5
penicillins in milk by hplc with pre-column
derivatization. J. Instrum. Anal., 19(6): 72-74.
Fig. 4: The standard curve of monoclonal antibody against
Ampicillin
polyclonal antibody in immune analysis of drug
residues (Tan and Shi, 2000).
In the ELISA reaction system, multiple factors can
affect the preparation of standard curve, including pH
value, ionic strength, organic solvent, reaction
temperature, reaction time and the amount of reactants
and sample pretreatment method. The hydrogen
bonding between antigen and antibody as well as
ionized antibody ligand are substantially affected by pH
values, which in turn affects the coating antigen and the
molecular structure of the sample. Within a certain
range of ionic strength, the high ionic strength can
reduce the adsorption of the non-specific antibody. This
is because the charge involved in the non-specific
reactions can be covered by the large number of ions.
However, if the ion concentration is too high, the
specific antigen-antibody reaction will be affected and
the detection sensitivity will decline greatly. The
organic solvent may destroy the van der Waals force
and the hydrophobic interaction between antigen and
antibody, thus dissociating the antigen-antibody
complex. In real practices, the pH value, ionic strength
and organic solvent in the reaction system are affected
by screening of various working fluids. In the present
786